Amycolatopsis sp. strain and methods of using the same for vanillin production

ABSTRACT

This invention provides an  Amycolatopsis  sp. strain (zhp06), and a method of using the whole cell preparation of the strain for vanillin production. The strain was deposited in China Center for Type Culture Collection on Jul. 26, 2011 with the number of CCTCC NO: M 2011265. Under high concentrations of ferulic acid substrate, the vanillin production by this method can reach more than 10 g/L. The molar conversion rate of ferulic acid is more than 50% and the purity of vanillin is from 80% to 95%. The advantage of this invention includes: repeated use of biocatalyst cells, mild biotransformation condition, low environmental pollution, short production cycle, high product purity and simple purification procedure. It has a great potential for industrial applications.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Continuation Application of U.S. Continuationapplication Ser. No. 14/800,261, filed Jul. 15, 2015, which is aContinuation Application of U.S. application Ser. No. 13/591,231, filedAug. 22, 2012, now U.S. Pat. No. 9,115,377, issued Aug. 25, 2015, whichclaims the benefit of Chinese Patent Application No. 201110325488.1,filed Oct. 24, 2011, the contents of each of which are incorporated byreference in their entirety herein.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on May 22, 2017, isnamed 070946-082563-C2_SL and is 904 bytes in size.

BACKGROUND OF THE INVENTION

Field of the Invention

This invention is in the field of biotechnology, and in particular, itrelates to a new Amycolatopsis sp. Strain (zhp6), and methods for usingthe new strain to produce vanillin from ferulic acid.

Description of the Related Art

Vanillin (chemical name: 3-methoxy-4-hydroxybenzaldehyde) has amolecular weight of 152.1. Vanillin appears as white to slightly yellowneedle-like crystal or crystal powder. It is the primary component ofthe extract of vanilla seed pods. Vanillin exists in natural plants infree or glucoside forms. It accounts for 1.5% to 3% of the dry weight ofvanilla beans, and is the major component among more than 200 flavorsfound in vanilla. Due to its creamy sweet flavor and aroma, vanillin iswidely used as a flavor compound in ice cream, chocolate and dairydesserts and other food products, and is called the world's most widelyused flavor enhancer.

In today's market, most vanillin is chemically synthesized from guaiacol(annual production of 12000 tons, $16/kg), and only very small portionof vanillin is naturally extracted from vanilla planifolia (annualproduction of 20 tons, $3200/kg). Although chemical synthesis to producevanillin is a mature technology, the method brings serious environmentalpollution. The security of chemical production of vanillin began to bequestioned. Another disadvantage of chemical method is that it producesa product with only single aroma. Due to the high production cost andhigh consumption price, naturally extracted vanillin cannot meetpeople's demand for consumption. This promotes the development ofbio-transformation technologies for vanillin production. In recentyears, it has become a current trend to produce bio-vanillin fromprecursor substrates using microbial cells (Current Opinion inBiotechnology 2000, 11:490-496).

Ferulic acid is a precursor substrate of vanillin production. Dependingon types of micro-organisms, there are one-step and two-step microbialtransformation methods for vanillin production. Chinese Patent Nos.CN1421523A and CN 1824783A disclosed a two-step method that ferulic acidis first converted to vanillic acid, and vanillic acid is converted tovanillin using Aspergillus niger CGMCC 0774 and PycnoporuscinnabarinusCGMCC 1115. This method requires the usage of two microbial organismsand is very time consuming.

U.S. Pat. No. 6,133,003 disclosed a one-step method for convertingferulic acid to vanillin using Amycolatopsis DSM 9991 and DSM 9992strains. After microbial cells grew in the fermenter for 12.5 hours,ferulic acid was added to fermentation broth at different stages.Fermentation continued for another 50 hours until vanillin infermentation broth reached 11.5 g/L with a converting rate of 77.8%.U.S. Pat. No. 6,235,507 disclosed a one-step method using Streptomycessetonni ATCC 39116. Microorganisms were inoculated in the growth mediumand grew for 20-40 hours. When glucose in the growth medium wasconsumed, ferulic acid substrate was added to the fermentation broth atdifferent phases. Fermentation was continued for another 5-50 hours. Thefermentation broth can accumulate 8-16 g/L of vanillin and by-productssuch as vanillic alcohol, vanillic acid, guaiacol, vinyl guaiacol and2-methoxy-4-ethyl phenol. Organic solvents such as methyl butyl ether(MTBE) was used to adjust pH and separate vanillin from the by-products.The yield of vanillin and the conversion rate in the above-mentionedinventions is relatively high, but the timing of adding ferulic acidsubstrate is hard to control, the microorganism strain is difficult toobtain, and there are many byproducts generated by these methods.Another method by Qingli Zhou used Streptomyces sp. L1936 strain forconverting ferulic acid to vanillin. This method needs to add ferulicacid two times to the fermentation broth. The yield of vanillin was 7.12g/L and the molar conversion rate was 69.9% (Food and FermentationIndustries, 2004, 30 (3): 18-20).

Chinese patent No. CN 101165168A disclosed a fermentation method usingStreptomyces sp. V-1 (CCTCC M 206065) strain for generating vanillinfrom ferulic acid. This method used CY growth medium for growingmicroorganisms and DM11 macroporous absorbent resin for vanillinpurification. Ferulic acid was added to the fermentation broth atdifferent stages until the final concentration reached 45 g/L. The finalyield of vanillin was 19.2 g/L and the molar conversion rate was 54.5%.The disadvantage of these two methods is that they both need to addferulic acid for multiple times, which makes the operational controldifficult.

SUMMARY OF THE INVENTION

This invention provides an Amycolatopsis sp. strain (zhp06) and methodsfor converting ferulic acid to vanillin using the Amycolatopsis sp.strain.

This invention provides an Amycolatopsis sp. strain (zhp06), which wasdeposited in China Center for Type Culture Collection on Jul. 26, 2011with the number of CCTCC NO: M 2011265. The characteristic of thisstrain is as follows. The body of the bacteria is filamentous, thecolony color is incanus or yellow, and the spore appears ellipsoid. Theoptimal temperature for growth is from 28° C. to 37° C. with the abilityto grow at 50-55° C. Its 16S rRNA sequence shares 98-99% homology withthose of many known Amycolatopsis sp. strains.

This invention provides a method of converting ferulic acid to vanillinusing strain Amycolatopsis sp. zhp06, comprising the following steps:

1. Amycolatopsis sp. zhp06 cells or mutant strains were cultivated usinga standard culture process. The cultured cells were collected bycentrifugation.

2. The cells obtained from step 1 were added to a substrate solution toform a biotransformation solution. The cells were used as a biocatalystto convert ferulic acid to vanillin during the biotransformationreaction.

3. Vanillin was extracted from the above biotransformation solutionusing macroporous absorbent resins.

In step one, the fermentation medium contains glucose 5-15 g/L, yeastextract 1-20 g/L, Na₂HPO₄.10H₂O 1-10 g/L, KH₂PO₄ 0.1-2 g/L, NaCl 0.1-0.5g/L, MgSO₄.7H₂O 0.2-0.5 g/L, CaCl₂.2H₂O 0.01-0.1 g/L, ferulic acid0.05-0.5 g/L, pH 7.2-7.4. The above medium is sterilized at 121° C. for20 min.

In step two, the ratio of wet weight of zhp 06 cells (g) and the volumeof the ferulic acid solution (ml) is from 1:2 to 1:20. The substratesolution contains ferulic acid 3-40 g/L, Na₂HPO₄.10H₂O 1-10 g/L andKH₂PO₄ 0.1-0.5 g/L (pH 7.5-7.9). The biotransformation reaction wasperformed at 28-45° C. for 20-70 hours.

After 5-12 hours of biotransformation reaction in step 2, 5-10% (w/v,g/ml) macroporous resin HZ-16 or HZ-802 (Shanghai Huazhen Science andTechnology Inc., Shanghai, China) was added to the biotransformationsolution. When the extracting reaction was over, the macroporous resinwas filtered out and eluted with acetic ether or alcohol (2 times of theresin volume) at 35-40° C. The eluted solution was dried by anhydroussodium sulfate for 12-15 hours and was filtered to remove sodiumsulfate. The filtered solution was concentrated to achieve 200-230 g/Lvanillin under vacuum evaporation. The concentrated solution was thenleft at 4° C. for vanillin crystallization.

The carbon source in fermentation medium is selected form glucose,starch, sucrose, fructose and maltose.

DETAILED DESCRIPTION

Screening and Identification of Microbial Strains

Actinobacteria were isolated from eleven soil samples collected fromWuxi and Kunming in China. The samples were diluted and plated on agarplates with 2,4-dinitrophenylhydrazine. The plates were incubated at 28°C. for 3-5 days. Colonies with red or orange transparent circle wereselected for second round screening. The selected cells were cultured inliquid fermentation medium for two to five days, after which cells in 5ml fermentation liquid were collected by centrifugation. Wet cells wereadded to substrate solution containing 5 g/L ferulic acid and incubatedat 37° C. for 24-48 hours to convert ferulic acid into vanillin. Theconversion products were further analyzed by silica gel chromatography(benzene:Hexane:chloroform:ether:acetic acid=4:3:2:1:0.1, v/v). Thestrain zhp06 was found to accumulate significant amount of vanillin.

Genomic DNA of strain zhp06 was extracted and its 16S rRNA sequence wasfound to have 98-99% similarity to those of many known Amycolatopsis sp.strains. According to its biochemical and physiological properties andstandards set in “Actinomycetes Systems” (Science Press, 2007, P363) and“Basis of Classification of Actinomycetes” (Science Press, 1977), thestrain was recognized as an Amycolatopsis sp. Zhp06 was then depositedin China Center for Type Culture Collection on Jul. 26, 2011 with thenumber of CCTCC NO: M 2011265.

Prominent features of this strain includes the following: a) the cellbody is filamentous, the colony color is incanus or yellow, and thespore appears ellipsoid; b) the optimal growth temperature is from 28°C. to 37° C. with the ability to grow at 50-55° C., the optimum growthpH for Zhp06 cells is pH 6-9, and the optimal growth NaCl concentrationis 0-5%; c) they can make milk coagulation and gelatin liquefaction, andproduce catalase, urease, and lipase. They cannot break down celluloseas the amylase activity in these cells is low; the cells cannot produceH₂S or acetylmethyl-methanol; they produce very little formic acid andother acidic substances when breaking down glucose; they can produceindole.

Cell Culture and Production of Vanillin

The slant medium contains glucose agar medium, asparagine agar medium orISP-2 medium. (“Catalog of Chinese Bacterial Strains”, Chemical IndustryPress, 2007)

The screening medium adds 0.5-5 g/L benzoic acid or ferulic acid to theslant medium.

The fermentation medium contains carbon source 5-15 g/L, yeast extract1-20 g/L, Na₂HPO₄.10H₂O 1-10 g/L, KH₂PO₄ 0.1-2 g/L, NaCl 0.1-0.5 g/L,MgSO₄.7H₂O 0.2-0.5 g/L, CaCl₂.2H₂O 0.01-0.1 g/L, ferulic acid 0.05-0.5g/L, (use NaOH to adjust the pH to 7.2-7.4). The above medium wassterilized at 121° C. for 20 minutes.

The carbon source in fermentation medium is selected from glucose,starch, sucrose, fructose and maltose.

After Amycolatopsis sp. zhp06 was cultivated on the agar slant at 28-37°C. for 3-6 days, the spores were suspended in sterile water and added tothe fermentation medium at the ratio of 2%-5% (v/v). Cells wereharvested by centrifugation (3000-6000 g, 10-15 min) after cultivationat 28-40° C., 180-220 rpm for 22-40 hours.

As a biocatalyst, the wet zhp06 cells or immobilized zhp06 cells wereadded to the substrate solution at the ratio from 1:2 to 1:20(weight/volume) to form a biotransformation solution. The substratesolution contains 3-40 g/L ferulic acid, 1-10 g/L Na₂HPO₄.10H₂O, 0.1-0.5g/L KH₂PO₄, pH 7.5-7.9. The biotransformation reaction was performed at28-45° C. for 20-70 hours.

The immobilized cells were prepared by using carrageenan, chitosan orcalcium alginate as the immobilization carrier (<<Immobilization ofEnzyme and Cell>>, Chemical Industry Press, 2006).

Purification of Product

After 5-12 hours of biotransformation reaction, macroporous resin HZ-16or HZ-802 was added into the biotransformation solution at a ratio of 5%to 50% (weight (g)/volume (ml)). When the reaction was over, themacroporous resin was filtered out and eluted at 35-40° C. with aceticether or alcohol (two times the wet volume of macroporous resin). Theeluted solution was dried by anhydrous sodium sulfate for 12-15 hours,and was filtered to remove sodium sulfate. The filtered solution wasconcentrated under vacuum evaporation to achieve 200-230 g/L vanillin.The concentrated vanillin solution was then left at 4° C. for vanillincrystallization.

Product Analysis and Quantification

Vanillin in biotransformation samples were extracted with butyl acetate(analytic grade) and analyzed by HPLC. (Determination of Vanillin,Eugenol and Isoeugenol by RP-HPLC. Chromatographia 60:709-713)

This invention provides an Amycolatopsis sp. strain (zhp06) with theability to convert high concentrations of ferulic acid to vanillin. Italso provides a method for vanillin production using the whole zhp06 andits mutant cells as a biocatalyst. The bacteria cells are first culturedunder standard culture conditions. The cells are then harvested and usedas a biocatalyst to convert high concentrations of ferulic acidsubstrate into vanillin. One advantage of the method is that thebiocatalyst, the Amycolatopsis sp. zhp06 cells can be used repeatedly.The reaction condition of the method is very mild, leading to verylittle environmental pollution. Another advantage is that the productioncycle of this method is short, and the purity of conversion product isvery high, making it easy to be purified. This method has greatpotential for industrial applications.

Bio-Sample Storage:

Amycolatopsis sp. zhp06 was deposited in China Center for Type CultureCollection on Jul. 26, 2011 with the number of CCTCC NO: M 2011265.

Examples Example 1. Isolation and Characterization of Zhp 06 Strain

Soil samples were taken from Wuxi and Kunming in China. The samples werediluted and plated onto agar plates with 2,4-dinitrophenylhydrazine. Theplates were incubated at 28° C. for 3-5 days and colonies with red ororange transparent circle were selected. Furthermore it was screened bythe method of silica gel chromatography(benzene:Hexane:chloroform:ether:acetic acid=4:3:2:1:0.1). The strainzhp06 was selected for its ability to produce high amounts of vanillin.

Genomic DNA of the strain zhp06 was extracted using a bacterial DNAextraction kit (Shanghai Generay Biotech Company, Shanghai, China). The16S rRNA gene of zhp06 was amplified by PCR using the universalbacterial RNA primers (upstream primer ACGGTTACCTTGTTACGACTT (SEQ ID NO.1), downstream primer AGAGTTTGATCCTGGCTCAG (SEQ ID NO. 2)). Sequencingof the 16S rRNA sequences was performed by Shanghai Sangon Co., Ltd. andthe sequences were submitted to GenBank with a registration number ofJF828149. NCBI-BLAST was used to search for sequence homology with other16S rRNA sequences in GenBank (see the result in Table 1). Thephysiological and biochemical properties of zhp06 strain wascharacterized as described in “Actinomycete Systematics”. A salientfeature of the strain is that it possesses high tolerance to changes inpH, temperature, and NaCl concentrations. Amycolatopsis eurythermastrain NT202 and Amycolatopsis thermoflava strain 173573 are twobacteria strains that have the highest sequence homology with zhp 06strain. They are similar to each other in terms of their tolerance tochanges in growth temperature and NaCl concentrations. But they usedifferent carbon sources (e.g. arabinose, cellobiose, xylose, etc.). Thezhp06 strain shares the same property with Amycolatopsis eurythermastrain NT202 in terms of gelatin digestion, and production of enzymessuch as amylase, nitrate reductase, and urease. Therefore, zhp06 strainis considered to be an Amycolatopsis sp. The strain was deposited intoChina Center for Type Culture Collection on Jul. 26, 2011 with thenumber of CCTCC NO: M 2011265.

TABLE 1 16S rRNA Sequence Homology Analysis Strain Name NCBI NO.Homology Amycolatopsis thermoflava strain 173573 EU570741.1 99%Amycolatopsis sp. ATCC 39116 AM263203.1 99% Amycolatopsis eurythermastrain NT202 NR_036887.1 98% Amycolatopsis thermoflava strain N1165NR_024890.1 98% Amycolatopsis methanolica AJ249135.1 98% Amycolatopsisthermoflava strain N1165 NR_024890.1 98% Amycolatopsis methanolicapartial X54274.1 97% Amycolatopsis albidoflavus AB327251.1 95%Amycolatopsis taiwanensis AB327255.1 94% Amycolatopsis orientalis IFO12806^(T) AJ400711 92% Streptomyces eurythermus D63870 90% Amycolatopsismediterranei DSM 13685^(T) X76957 90% Streptomyces setonii NBRC 13085AB184300.1 89% Pseudonocardia thermophila ATCC 19285T X53195 86%Bacillus subtilis subsp AL009126.3 77% Bacillus fusiformis AM062692.173%

TABLE 2 Biochemical and physiological properties of Amycolatopsis sp.zhp06 Characteristic zhp06 1 2 Aerial mycelium + + + Color of aerialmycelium white white white Utilization of: L-(+)-Arabinose w + +D(+)-Cellobiose w + + Dextrin/dextran w + − D-(+)-Fructose + + +D-(+)-Glactose w + + meso-ino-sitol − + − D-(+)-lactose w w +D-(+)-maltose w − − D-(+)-Mannitol w + + D-(+)-Melezitose − o −D-(−)-Sorbitol − + + sucrose − − − D-(+)-Trehalose − + + D-(+)-Xylosew + + Hydrolysis of: Gelatin + + − Growth Growth at 10° C. − − − Growthat 45° C. + + + NaCL(5%) + + + Product o Amylase − − Nitratereductase + + Urease + + 1, stands for Amycolatopsis eurytherma strainNT202; 2, stands for Amycolatopsis thermoflava strain 173573; +, meanspositive; w, means slightly positive; −, means negative.

Example 2. Use Different Carbon Sources for Culturing Amycolatopsis sp.Zhp06 Cells

The spore of Amycolatopsis sp. zhp06 was inoculated to fermentationmedium with different carbon sources selected from maltose, fructose,glucose, soluble starch or sucrose. The zhp06 cells grew at 35° C., 180rpm for 24 hours. 20 mL fermentation broth was centrifuged at the speedof 5000 rpm for 10 minutes, and the cells were collected. The cells wereadded into substrate solution with 12 g/L ferulic acid. Thebiotransformation reaction was performed at 35° C., 180 rpm for 48hours.

The fermentation medium contained 5 g/L carbon source, 10 g/L yeastpowder, 5 g/L Na₂HPO₄.10H₂O, 0.5 g/L KH₂PO₄, 0.2 g/L NaCl, 0.2 g/LMgSO₄.7H₂O, 0.1 g/L CaCl₂.2H₂O, 0.1 g/L ferulic acid (pH 7.4). The abovemedium was sterilized at 121° C. for 20 min. Carbon source mentionedabove was selected from maltose, fructose, glucose, soluble starch andsucrose. The production of vanillin and other related compounds by zhp06cells cultured in different carbon sources was compared and shown intable 3.

TABLE 3 Products of Amycolatopsis sp. zhp06 utilizing different carbonsources Ferulic Vanillic Vanillyl Yield of Carbon acid acid GuaiacolVanillin alcohol vanillin source (g/L) (g/L) (g/L) (g/L) (g/L) (%)maltose 1.49 0.82 0.55 5.08 0.40 54.1 fructose 2.02 0.39 0.25 5.48 0.6658.3 glucose 1.91 0.64 0.27 4.96 0.64 52.7 Soluble 1.34 0.90 0.33 5.310.63 56.5 starch Sucrose 1.56 0.73 0.29 5.06 0.51 53.8

Example 3. Repeated Use of Biocatalyst Cells

The cultivation method was as Example 2 with glucose as the carbonsource, 100 ml fermentation broth was centrifuged for 10 minutes at thespeed of 5000 rpm, and then the cells were collected. 3.5 g cells wereadded into 20 ml substrate solution with 16 g/L ferulic acid. Thebiotransformation reaction was performed at 35° C., 180 rpm, for 24hours. After biotransformation, the cells were recycled and added intoanother 20 mL substrate solution with 16 g/L ferulic acid again. Thecells were repeatedly used for 7 times and the results were shown inTable 4.

TABLE 4 Analysis for products generated by recycled cells VanillylVanillic Ferulic Yield of Repeat alcohol Vanillin acid Guaiacol acidVanillin times (g/L) (g/L) (g/L) (g/L) (g/L) (%) 1 0.34 3.51 0.76 0.565.6 28 2 0.55 3.82 3.64 1.58 0.04 30 3 0.94 7.97 1.81 0.46 0.14 64 40.95 7.41 1.30 0.40 0.36 59 5 0.79 8.02 1.40 0.24 0.76 64 6 0.87 8.211.85 0.09 0.23 65 7 1.39 7.93 3.25 0.15 1.17 63

Example 4. Process of Making Immobilized Cells

The immobilized cells were obtained by the following method:

1) 5 g cells were obtained by the method as described in example 2;

2) The cells in step 1) were suspended in the same volume of saline andmixed with 5.4% carrageenan solution at the ratio of 1:2 (v/v) at 58°C.;

3) The mixture in step 2) was immersed in 0.3M cold potassium chloridesolution and reacted at 10° C. for 4 hours.

10 g immobilized cells were added into substrate solution with 16 g/Lferulic acid. The biotransformation reaction was performed at 37° C. for24 hours. The concentration of vanillin was measured and the immobilizedcells were recycled. The above steps were conducted repeatedly for 3times, and the concentration of vanillin achieved was 5.6 g/L, 5.3 g/L,4.4 g/L, respectively.

Example 5. Biotransformation Reaction with Different SubstrateConcentrations

5 g cells (obtained from example 2 using starch as the carbon source)were added into 20 ml substrate solution with 20, 30, and 40 g/l ferulicacid. The biotransformation was performed at 35° C., 180 rpm for 12hours. 2 g macroporous resin HZ-16 was then added to thebiotransformation solution, and was incubated at 35° C. for another 48hours. Production of vanillin was shown in Table 5.

TABLE 5 Effect of substrate concentrations on production of vanillinsubstrate Vanillyl Vanillic concentration alcohol Vanillin acid GuaiacolFerulic (g/L) (g/L) (g/L) (g/L) (g/L) acid (g/L) 20 0.61 7.88 0.03 6.460.46 30 0.19 10.03 0.06 5.51 0.65 40 0.46 10.08 2.16 4.52

Example 6. Purification of Vanillin

The purification method comprises the following steps:

-   -   1) 1 L biotransformation solution containing 5.34 g/L Vanillin        were added to 300 g macroporous resin HZ-16. After 3 hours of        absorption, macroporous resin HZ-16 were filtered out and eluted        twice with 600 ml acetic ether at 35° C., each time for 2 hours.    -   2) The acetic ether elute (˜1200 ml) was dried by anhydrous        sodium sulfate for 12-15 hours and was vacuum evaporated until        vanillin concentration reaching 22%.    -   3) The solution obtained from step 2 was settled at 4° C. for        crystallization.    -   4) 3.66 g vanillin was obtained with purity of 95%.

While the present invention has been described in some detail forpurposes of clarity and understanding, one skilled in the art willappreciate that various changes in form and detail can be made withoutdeparting from the true scope of the invention. All figures, tables,appendices, patents, patent applications and publications, referred toabove, are hereby incorporated by reference.

What is claimed is:
 1. A method for producing vanillin comprising: a.adding Amycolatopsis sp. zhp06 cells isolated from a standard cellculture to a substrate solution comprising about 3-40 g/L ferulic acidto form a biotransformation solution, whereby the ferulic acid isconverted to vanillin using said Amycolatopsis sp. zhp06 cells as abiocatalyst; b. after said conversion of ferulic acid to vanillin,extracting the vanillin from the biotransformation solution to produce aconcentrated extract containing 200-230 g/L of vanillin, wherein theextracting is performed by adding 5-50% (w/v) of macroporous absorbentresin to said biotransformation solution and then eluting the vanillinfrom the resin; and c. crystallizing the vanillin from the concentratedextract thereby producing purified vanillin using said Amycolatopsis sp.zhp06 cells.
 2. The method of claim 1, wherein the standard cell culturecomprises a fermentation medium containing 5-15 g/L carbon source, 1-20g/L yeast powder, 1-10 g/L Na₂HPO₄.10H₂O, 0.1-2 g/L KH₂PO₄, 0.1-0.5 g/LNaCl, 0.2-0.5 g/L MgSO₄.7H₂O, 0.01-0.1 g/L CaCl₂.2H₂O, and 0.05-0.5 g/Lferulic acid.
 3. The method of claim 2, wherein the carbon source isselected from glucose, starch, sucrose, fructose and maltose.
 4. Themethod of claim 2, wherein said fermentation medium is sterilized at121° C. for 20 min.
 5. The method of claim 1, wherein said substratesolution contains 3-40 g/L ferulic acid, 1-10 g/L Na₂HPO₄.10H₂O, and0.1-0.5 g/L KH₂PO₄ having pH 7.5-7.9.
 6. The method of claim 1, whereinsaid conversion of ferulic acid to vanillin is performed at 28-45° C.for 20-70 hours.
 7. The method of claim 1, wherein a weight-to-volumeratio (w/v) of the wet weight (g) of said Amycolatopsis sp. zhp06 cellsand the volume (ml) of said substrate solution is from 1:2 to 1:20. 8.The method of claim 1, wherein after 5-12 hours of said conversion offerulic acid to vanillin, the vanillin is extracted and eluted from theresin with acetic ether or alcohol to, and wherein the eluent is driedto produce the concentrated extract.
 9. The method of claim 8, whereinthe eluent is dried by anhydrous sodium sulfate and is vacuum evaporatedto produce the concentrated extract.
 10. The method of claim 8, whereinthe macroporous absorbent resin is HZ-16 or HZ-802.
 11. The method ofclaim 10, wherein said eluent is dried by anhydrous sodium sulfate for12-15 hours.
 12. The method of claim 1, further comprising recyclingsaid Amycolatopsis sp. zhp06 cells from the biotransformation solutionand adding said cells into a fresh biotransformation solution comprisingthe ferulic acid for another vanillin production.
 13. The method ofclaim 1, wherein said Amycolatopsis sp. zhp06 cells is isolated from thestandard cell culture by centrifugation.
 14. A method for producingvanillin comprising: a. adding Amycolatopsis sp. zhp06 cells to asubstrate solution comprising about 3-40 g/L ferulic acid to form afirst biotransformation solution, whereby the ferulic acid is convertedto vanillin using said Amycolatopsis sp. zhp06 cells as a biocatalyst;b. after said conversion reaction of ferulic acid to vanillin, recyclingsaid Amycolatopsis sp. zhp06 cells from the first biotransformationsolution; and c. adding the recycled Amycolatopsis sp. zhp06 cells intoa second biotransformation solution comprising ferulic acid for anothervanillin production.